AMB Express
Characterization of the depolymerizing activity of commercial lipases and detection of lipase-like activities in animal organ extracts using poly(3-hydroxybutyrate-co-4-hydroxybutyrate) thin film
Pei-Shze Moka, Diana Hooi-Ean Ch’nga, Soo-Peng Onga, Keiji Numatab and Kumar Sudesh*a
a School of Biological Sciences, Universiti Sains Malaysia, 11800 Pulau Pinang, Malaysia.
b Enzyme Research Team, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
* Corresponding author
Mailing address:
Kumar Sudesh
School of Biological Sciences,
Universiti Sains Malaysia,
11800 Penang,
Malaysia.
Tel: +60-4-6534367
Fax: +60-4-6565125
E-mail:
Table S1 Physical and thermal properties of P(3HB-co-4HB) (Saito et al., 1996)
4HB fraction (mol %)0 / 3 / 7 / 10 / 16 / 27 / 64 / 78 / 82 / 90 / 100
Melting temp (°C) / 178 / 172 / 130 / 50 / 49 / 52 / 50 / 53
Glass transition temp (°C) / 4 / −2 / −7 / −35 / −37 / −39 / −42 / −48
Crystallinity (%) / 60 / 55 / 50 / 45 / 45 / 40 / 15 / 17 / 18 / 28 / 34
Density (g/cm3) / 1.250 / 1.232 / 1.234 / 1.234
Water uptake (wt %) / 0.32 / 0.20 / 0.14 / 0.45
Stress at yield (MPa) / 34 / 28 / 19 / 14
Elongation at yield (%) / 4 / 5 / 7 / 17
Tensile strength (MPa) / 43 / 28 / 24 / 26 / 17 / 42 / 58 / 65 / 104
Elongation to break (%) / 5 / 45 / 242 / 444 / 591 / 1120 / 1320 / 1080 / 1000
Table S2 Sources and concentration used of commercial lipases (Sigma-Aldrich technical information)
Lipase name / Product number / Lipase activity (U/mg) / Unit definition / Concentration used in this study (mg/mL)Lipase from Candida antarctica / 65986 / ≥1.0 / 1 U corresponds to the amount of enzyme which liberates 1 μmol oleic acid per min at pH 8.0 and 40 °C (triolein, Fluka No. 62314 as substrate) / 0.05
Lipase from Candida rugosa, Type VII / L1754 / ≥700 / 1 U will hydrolyze 1.0 microequivalent of fatty acid from a triglyceride in 1 h at pH 7.2 at 37 °C / 1.00
Amano Lipase M from Mucor javanicus / 534803 / ≥10 / - / 0.50
Lipase from porcine pancreas, Type II / L3126 / 100-400 (using olive oil, 30 min incubation), 30-90 (using triacetin) / 1 U will hydrolyze 1.0 microequivalent of fatty acid from triacetin in 1 h at pH 7.4 at 37 °C. (pH 7.7 is used with olive oil as substrate) / 0.25
Lipase from Pseudomonas cepacia / 62309 / ≥30 / 1 U corresponds to the amount of enzyme which liberates 1 μmol oleic acid per min at pH 8.0 and 40 °C (triolein, Fluka No. 62314 as substrate) / 0.25
Amano Lipase from
Pseudomonas fluorescens / 534730 / ≥20 / - / 1.00
Lipase from Rhizopus arrhizus / 62305 / ~10 / 1 U corresponds to the amount of enzyme which liberates 1 μmol of butyric acid per minute at pH 8.0 and 40 °C (tributyrin, Fluka No. 91010 as substrate); 5000 U as described above are equivalent to ~1 U using triolein, Fluka No. 62314 as substrate, at pH 8.0 and 40 °C / 0.12
Lipase from Rhizopus niveus / 62310 / ≥1.5 / 1 U corresponds to the amount of enzyme which liberates 1 μmol fatty acid from a triglyceride per minute at pH 7.7 and 40 °C (olive oil as substrate)]; 300 U as described above are equivalent to ~1 U using triolein, Fluka No. 62314, at pH 8.0 and 40 °C as substrate / 4.00
Lipase from Rhizopus oryzae / 80612 / ≥30 / 1 U corresponds to the amount of enzyme which releases 1 μmol fatty acid from triglycerides per minute at pH 7.2 and 37 °C (olive oil as substrate) / 0.25
Table S3 Compounds used for buffers preparation with different pH
pH / Compounds / Volume (mL)1 / 0.2 M KCl / 5.000
0.2 M HCl / 13.400
2 / 0.2 M KCl / 5.000
0.2 M HCl / 1.300
3 / 0.2 M Na2HPO4 / 2.040
0.1 M citric acid / 7.960
4 / 0.2 M Na2HPO4 / 3.860
0.1 M citric acid / 6.140
5 / 0.2 M Na2HPO4 / 5.140
0.1 M citric acid / 4.860
6 / 1 M K2HPO4 / 0.264
1 M KH2PO4 / 0.770
7 / 1 M K2HPO4 / 1.230
1 M KH2PO4 / 0.770
8 / 1 M K2HPO4 / 1.880
1 M KH2PO4 / 0.120
9 / 0.2 M glycine / 5.000
0.2 M NaOH / 0.880
10 / 0.05 M NaHCO3 / 10.000
0.1 M NaOH / 2.140
11 / 0.05 M NaHCO3 / 10.000
0.1 M NaOH / 4.540
12 / 0.05 M NaHCO3 / 10.000
0.1 M NaOH / 5.380
13 / 0.2 M KCl / 5.000
0.2 M NaOH / 13.200
The solutions were topped up to 20 mL.
Figure captions
Fig. S1 Schematic diagram of degradation of amorphous part of P(3HB-co-4HB) film
Fig. S2 Relationship of weight loss of P(3HB-co-4HB) film and relative density of opaque spot on P(3HB-co-4HB) film caused by lipase from a) P. fluorescens and b) C. rugosa
Fig. S3 Hydrolysis spots of PBS pH 7.4 on P(3HB-co-4HB) film under different temperature from 15 °C to 60 °C. The assay was conducted for 30 minutes.
Hydrolysis spots were observed at 50, 55 and 60 °C indicating the occurrence of non-enzymatic hydrolysis
Fig. S4 Depolymerizing activity assay on P(3HB-co-4HB) by extract from mice organs which are (b) duodenum, (c) duodenum*, (d) liver, (e) liver*, (f) spleen, (g) spleen*, (h) heart, (i) heart*, (l) pancreas, (m) pancreas*, (n) stomach, (o) stomach*, (p) lungs, (q) lungs*, (r) kidney, (s) kidney*. (a) and (k) indicate 0.25 mg/mL of lipase from P. cepacia which act as positive control while (j) and (t) indicate PBS which act as negative control. The assay was carried out at 37 °C for 1 hour. Each row of spots indicates triplicate. * indicates supernatant heated at 95 °C for 30 minutes
Fig. S5 Depolymerizing activity assay on P(3HB-co-4HB) by extract from chicken organs which are (c) duodenum and pancreas, (d) duodenum and pancreas*, (e) fat, (f) fat*, (g) gizzard, (h) gizzard*, (k) liver, (l) liver*, (m) large intestine, (n) large intestine*, (o) small intestine, (p) small intestine*. (a) and (i) indicate 0.25 mg/mL of lipase from P. cepacia which act as positive control while (b) and (j) indicate PBS pH 7.4 which act as negative control. The assay was carried out at 37 °C for 1 hour. Each row of spots indicates triplicate. * indicates supernatant heated at 95 °C for 30 minutes
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